Lysis, extraction and purification of adeno-associated virus and adenovirus from host cells

ABSTRACT

The present disclosure provides compositions and methods for extracting viral particles of a recombinant adeno-associated virus (AAV) or an adenovirus (AdV) from a sample comprising cells enclosing the viral particles. The method can include lysing the cells with the addition of an alkaline lysis solution to the sample such that the sample comprises compound(s) buffering pH value about 9.0 to about 11.5 and about 0.1% to about 1% of a detergent precipitating host cell proteins by adding a salting solution to the sample such that the sample comprises about 0.5 M to about 2.0 M of a salt and has a pH of about 4.0 to about 7.0.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 6, 2017, is named52VP-246742-US_SL.txt and is 1,375 bytes in size.

BACKGROUND

Since “virus-like” particles were first discovered in adenovirus (AdV)preparations, adeno-associated virus (AAV) has been characterized anddeveloped in the last 50 years as a potent viral vector to deliver genesin vitro in cultured cells and in vivo in animal models. AAV is a small,“naked” virus containing a single-stranded DNA genome of approximately4.7 kb, consisting of two inverted terminal repeats (ITRs) that arecapable of forming T-shape secondary structure and acting as origins ofgenome replication, one rep region that encodes four overlappingreplication proteins, Rep78, Rep68, Rep52, and Rep40, and one cap regionthat encodes three structural proteins, VP1, VP2, and VP3. Naturallyisolated serotypes 1-9 of the AAV viruses share the genomic structurealthough these serotypes may display different tissue tropism. Numerousinvestigations have revealed their attractive features includingnonpathogenicity, efficient transduction and stable expression, thuslaying foundations for recombinant AAV vectors for use as one of themost successful gene delivery vehicles.

Production of recombinant AAV vectors (rAAV) is traditionally achievedby transfection of human-derived HEK293 cells with a rAAV viral vectorand a packaging construct in the presence of auxiliary viruses such asadenoviruses (AdV) that provide the helper function. Afteridentification of AdV regions required for AAV vector packaging, ahelper virus-free method was established using a constructed helperplasmid acting as auxiliary viruses. Therefore, a helper-free system isa triple transfection protocol consisting of three plasmids, whichsystem is widely used in research and drug development. In addition,development of baculovirus expression vectors provides another method toproduce rAAV viruses in insect sf9 cells. These different technologiesare shown to be able to produce sufficient quantities of rAAV virusesfor use in laboratories and clinical trials.

For purification of all of the different AAV serotypes, the viralparticles are first released from the packaging cells using 3-4freeze/thaw cycles, a conventional extraction method. In mostpurification protocols established to date, cesium chloride-(CsCl) andiodixanol-based density gradient ultracentrifugation is a central step.Although the gradient purification is useful in laboratory settings, ithas raised concerns for clinical applications. Such concerns, ingeneral, relate to (1) significant reduction of infectivity, (2)gradient-associated health risks, (3) gradient-associated toxicity, and(4) cumbersome procedures.

More recently, affinity and ion exchange chromatography have been testedfor AAV particle purification. Such technologies, however, areassociated with high cost and low scalability, or suffer fromdifficulties in separating viral particles from empty particles. Eitheraffinity or ion exchange chromatography alone is unable to produce highpurity of AAV viruses.

SUMMARY

The present disclosure, in certain embodiments, provides methods tolyse, extract and purify AAV or AdV viral particles from host cells. Themethods entail lysing the cells with an alkaline lysis solutioncomprising pH-buffering compound(s) or reagent(s) and a preferably milddetergent, followed by precipitating and removing the majority of hostcell proteins with a salting solution which preferably comprises a salt,under a relatively acidic condition. The extracted viral solution can bedesalted and enriched with certain polyethylene glycol (PEG) and furtherpurified by chromatography.

In accordance with one embodiment of the present disclosure, provided isa method of extracting viral particles of a recombinant adeno-associatedvirus (AAV) or an adenovirus (AdV) from a sample comprising cellsenclosing the viral particles. In some embodiments, the method compriseslysing the cells by the addition of an alkaline lysis solution to thesample such that the sample comprises about 0.1% to about 1% of adetergent and has a pH of about 9.0 to about 11.5. In one embodiment,the method further comprises precipitating host cell proteins by addinga salting solution to the sample such that the sample comprises about0.5 M to about 2.0 M of a salt and has a pH of about 4.0 to about 7.0.

In some embodiments, the viral particles are of any one of AAV serotypes1-11 or any one of AdV species A-G. In some embodiments, the detergentis a mild detergent. In some embodiments, the detergent is selected fromthe group consisting of deoxycholate, octyl thioglucoside, octylglucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside,alkyl Sulfates, Polysorbate 20 (Tween-20), Tergitol-type NP-40 and thecombinations thereof. In some embodiments, the detergent isdeoxycholate. In some embodiments, the detergent is deoxycholate andsodium dodecyl sulfate (SDS).

In some embodiments, the alkaline lysis solution has a pH of about 9.0to about 11.5 and comprises, in addition to the detergent, one or morereagents selected from the group consisting ofN-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine/sodium,glycine/potassium, sodium carbonate, potassium carbonate, sodiumphosphate, potassium-phosphate and combinations thereof. In someembodiments, the sample, following addition of the alkaline lysissolution, comprises about 0.1% to about 1.0% of the detergent.

In some embodiments, the salt is selected from the group consisting ofsodium acetate, potassium acetate, sodium phosphate and combinationsthereof. In some embodiments, the salting solution does not include anammonium salt.

In some embodiments, the method further comprises precipitating theviral particles by adding a precipitation solution to the sample suchthat the sample comprises about 5% to about 12% of a polyethylene glycol(PEG). In some embodiments, the PEG has an average molecular weight ofabout 3000 to about 10000.

In some embodiments, the method further comprises subjecting theprecipitated viral particles to chromatography. In some embodiments, thechromatography is silica-based chromatography, resin chromatography orion exchange chromatography columns.

In some embodiments, the method does not include the use of an organicsolvent. In some embodiments, the method does not include the use ofultracentrifugation. In some embodiments, the method does not includeextraction by freeze-thaw cycles. In some embodiments, the method doesnot include the use of affinity chromatography.

Also provided are formulations and compositions comprisingadeno-associated viruses (AAV) or recombinant adenoviruses (AdV)purified with any method of the above embodiments.

Provided in another embodiment of the present disclosure is apreparation of viral particles of a recombinant adeno-associated virus(rAAV) or an recombinant adenovirus (rAdV), extracted and purified froma sample of cells enclosing the viral particles, comprising at least1013 of the particles per milliliter (ml), wherein the preparationcontains at least 1010 transducing units (TU) per 1013 of the particles;contains less than 1/10,000 host cell proteins relative to the particles(w/w); and does not contain detectable cesium chloride (CsCl) oriodixanol. In some embodiments, no more than 1/10,000 of the viralparticles are empty particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 with panels A-D presents data demonstrating the tolerance ofadeno-associated virus (AAV) and adenovirus (AdV) to ranged pH valuesand to detergents to establish the foundation of development of lysissolutions used for lysis of their packaging cells. (A, B) AAV virus waspackaged by transfection of GFP-containing AAV viral vector andpackaging plasmids into HEK293 cells. GFP-containing AdV virus wasamplified by infection of HEK293 cells. Lentivirus (LtV) was packaged bytransfection of GFP-containing lentiviral vector and packaging plasmidsinto 293T cells, used for comparison. The viruses used here all werecollected from cultured medium of their packaging cells using 8%PEG8000, suspended in PBS and mixed with an equal volume of the pHsolutions as indicated. After 2 hours incubation at room temperature,the viral mixtures were used for infection in HEK293 cells.GFP-expressed cells were photographed and counted 3-4 days postinfection. Images of GFP cells shown are representatives of photos (A).GFP cell numbers were analyzed and shown in the scatter chart that isexpressed as mean percentage±SD of GFP cell numbers, n=6 (B). (C, D) AAVor AdV viruses were prepared as described above, mixed with an equalvolume of different concentrations of detergents, sodium dodecyl sulfate(SDS) or sodium deoxycholate (DOC), as indicated and incubated for 2hours at room temperature before infection as described above, n=5.

FIG. 2 with panels A-B shows that AAV and AdV particles are completelyreleased after lysis of their packaged cells using various alkalinesolutions with an appropriate amount of detergent. Seven alkaline lysissolutions were prepared according to the tolerance of AAV and AdV to pHvalues and detergents demonstrated in FIG. 1 and used for lysis of AAV-or AdV-packaged HEK293 cells as indicated. The lysates were used forinfection in HEK293 cells as described in FIG. 1 and the viral extractsfrom AAV- or AdV-packaged cells using freeze/thaw (F/T) 3 times wereused as comparative control. (A) Images of GFP cells shown arerepresentatives of photos and (B) histograms are expressed as meanpercentage±SD of GFP cell numbers. *, p<0.05; **, p<0.01, compared tothat of freeze/thaw extracted viruses (control), n=4.

FIG. 3 with panels A-C shows that AAV and AdV viruses exhibit hightolerance to a range of high concentrations of salt that enablessuccessful application of salting precipitation in purification of theseviral particles. (A) AAV or AdV viruses were prepared as described inFIG. 1 and mixed with an equal volume of different concentrations ofNaCl solutions as indicated. LtV virus was prepared and used forcomparison in parallel. After 2 hours at room temperature, viralmixtures were used for infection as described in FIG. 1. The scatterchart is expressed as mean percentage±SD of GFP cell numbers, n=4. (B,C) AAV- or AdV-packaged cells were lysed using 7 alkaline lysissolutions as described in FIG. 2 and a salt solution of 3 M NaAc, pH 5.5was added to each lysate for salting precipitation. After mixed,precipitates occurred and were removed by centrifugation. Supernatantswere saved for infection as described in FIG. 1. Images shown arerepresentatives of photos (B) and histograms are expressed as meanpercentage±SD of GFP cell numbers (C). *, p<0.05; **, p<0.01, comparedto that of freeze/thaw viruses (control), n=4.

FIG. 4 with panels A-C shows that salting precipitation removes themajority of protein impurities from alkaline lysates of AAV- andAdV-packaged cells in a salt dose-dependent manner. (A, B) Normal HEK293cells (Set 1) or AAV-packaged HEK293 cells (Set2) were lysed using 7alkaline lysis solutions as described in FIG. 2, followed by saltingprecipitation process as described in FIG. 3. The extracted samples wereused for BCA protein assay (A) or SDS polyacrylamide gel electrophoresis(PAGE) analysis (B). In parallel, extracts by freeze/thaw (F/T) 3 timeswere prepared as comparative control and cell lysates using RIPA bufferwas prepared as total protein control. Results from BCA assay areexpressed as mean percentage±SD of protein amount, **p<0.01, compared tothat of freeze/thaw viruses (control), n=6 (A). Results from PAGEanalyses are shown as images that are representatives of PAGE gels andexpressed as mean percentage±SD of staining density, *, p<0.05; **,p<0.01, compared to that of freeze/thaw viruses (control), n=3 (B). (C)AAV viruses or AdV viruses were packaged and lysed with a solution ofGlycine/Na, pH 10.0 and processed for salting precipitation usingdifferent amounts of NaAc (pH5.0 or 5.5) or KAc (pH5.0 or 5.5) asindicated. Extracted viral solutions were used for BCA assay. Scattercharts are expressed as mean percentage±SD of as protein amount, n=5.

FIG. 5 with panels A-D shows that PEG efficiently fractionates viralparticles into precipitates from extracted viral solutions after saltingprocess resulting in viral particles desalted, enriched and furtherpurified. AAV- or AdV-packaged cells were lysed with Glycine/Na, pH10.0, followed by salting process as described in FIG. 3. The extractedviral solutions were mixed with different amounts of PEG8000 asindicated. Precipitates after PEG fractionation were suspended in PBSand used for infection or BCA assay. (A) Results from infection areshown as images that are representatives of photos. (B) Results frominfection are shown as in histograms that are expressed as meanpercentage±SD of GFP cell numbers. *, p<0.05; **, p<0.01, compared tothat of freeze/thaw viruses (control), n=6. (C) Results from infectionare shown as images that are representatives of photos, whichdemonstrate that PEG efficiently fractionates AAV and AdV particles intoprecipitates. (D) Results from BCA assay are shown in a scatter chartthat are expressed as mean percentage±SD of protein amount, n=5.

FIG. 6 with panels A-C shows that silica-based chromatography as exampleis capable to further purify AAV and AdV viral particles. (A) AAVparticles or AdV particles were extracted using alkaline lysis/saltingprecipitation as described in FIG. 3 and the extracted viral solutionswere further purified using silica-based chromatography columns. (B) AAVparticles or AdV particles were PEG precipitated following alkalinelysis/salting precipitation processes, resuspended in an appropriatesolution and applied to silica-based chromatography columns. (C) AAV orAdV solutions eluted from columns as described in panels A and B wereconcentrated using Amicon Centrifugal Filter (Ultrcel-100K). Afterconcentration, the resulting viral solutions were titrated by means ofqPCR. Titers of these viral solutions were calculated as shown inhistograms that are expressed as mean±SD of viral genomic copies (GC)per ml, n=4.

FIG. 7 with panels A-G shows that purification of AAV as exampledemonstrates that the present approach yields a high-titer, ultrapureviral solutions. AAV-packaged cells were lysed with RIPA buffer (RIPA)as total protein sample or by freeze/thaw (F/T) as comparative controlor extracted using alkaline lysis/salting precipitation process(Extract) that was then PEG fractionated resulting in a suspension ofPEG precipitate (PEG_sus) as described in FIG. 5. The viral suspensionwas applied onto a silica-based chromatography column and the eluent wasconcentrated using a centrifugal filter to yield the resulting solution(Column) as described in FIG. 6. These samples were analyzed by PAGE gelelectrophoresis (A, C), BCA assay (B, D) or infection in HEK293 cells(E, F) as indicated. The purity of AAV viral solutions was calculated toexpress as viral transducing units (TU) per pg protein (G). (A-D)Results from PAGE gel analysis are shown as images that arerepresentatives of gels (A, C) and results from BCA assay are shown ashistograms that are expressed as mean percentage±SD of protein amount,n=5 (B, D). (E-G) Results from infection are shown as histograms thatare expressed as mean percentage±SD of GFP cell numbers (E, n=6), asmean±SD of transducing units (F, n=6) or as mean±SD of viral particlenumbers per pg protein (G, n=6).

FIG. 8 with panels A-F shows that the present approach with thecharacteristics to yields high recovery and purity of AAV viral solutionas example is unparalleled in the commercial market. AAV packaged cellswere collected and equally divided into 4 portions that were used forAAV purification by means of 4 different procedures, 3 commercial kits(marked as Kit A, Kit B and Kit C) that were available from differentcompanies and our above procedure (marked as C&M) as described in FIG.7. Kit A, Kit B and Kit C were employed following their productprotocols and the C&M procedure included alkaline lysis, saltingprocess, PEG fractionation and a silica-based chromatography column. Allpurified viral solutions were concentrated using Amicon Ultra-4 columnfilter to yield the resulting solutions in same volumes in PBS, followedby infection in HEK293 cells (A-C), PAGE gel electrophoresis (D) and BCAassay (E). Results from infection are shown as images that arerepresentatives of photos (A). Viral recovery and titer were calculatedas shown in histograms that are expressed as mean percentage±SD of GFPcell numbers (B, n=6) and as mean±SD of transducing units (C, n=6)respectively. Results from PAGE gel analysis are shown as images thatare representatives of gels (D) and results from BCA assay are shown inhistogram that is expressed as mean percentage±SD of protein amount (E,n=6). The purity of AAV viral solutions was calculated to express asmean±SD of transducing units (TU) per pg protein (F, n=6).

FIG. 9 shows the schematic diagram that outlines the example steps of anapproach of one embodiment of the disclosure for purification of AAV andAdV particles. These techniques are suitable for purification of AAV,AdV and any other “naked” viral particles that are packaged within thehost cells. The purification procedure includes four primary steps asindicated that can be finished within 3 hours or less: (1) Lysis of theviral packaged cells to release viral particles using an alkaline lysissolution with pH9.0-11.5; (2) Salting precipitation process to removethe majority of host cell protein impurities and retain viral particlesin solution using an salting solution; (3) PEG fractionation to separateviral particles into precipitates resulting in desalting, enrichment andfurther purification of viral particles; (4) Column chromatography tofinally purify viral particles, followed by concentration usingcentrifugal filters, to yield high-titer, ultrapure viral particles.These technologies do not use any toxic compounds for the viralpurifications so that the resulting viral solutions can meet the qualityrequirements for most research purposes and preclinical and clinicaltrials.

DETAILED DESCRIPTION Definitions

The following description sets forth exemplary embodiments of thepresent technology. It should be recognized, however, that suchdescription is not intended as a limitation on the scope of the presentdisclosure but is instead provided as a description of exemplaryembodiments.

As used in the present specification, the following words, phrases andsymbols are generally intended to have the meanings as set forth below,except to the extent that the context in which they are used indicatesotherwise.

Conventional methods for the extraction and purification of AAV and AdVparticles entail the use of freeze/thaw cycles followed with densitygradient ultracentrifugation. In some methods, affinity or ion exchangechromatography is not employed to improve purity of the final product.Such methods typically result in reduced infectivity of the viruses,accompanied by poor recovery. Moreover, for preclinical or clinical useof the viruses, the gradient-associated health toxicity can be asignificant concern.

One embodiment of the present disclosure provides a method of extractingand purifying AAV and AdV particles with improved recovery of the viralparticles with greater infectivity. Also, in this method, thefreeze/thaw cycles and density gradient ultracentrifugation are notrequired. The new method is easier to implement and readily scalable. Asfurther detailed below, one embodiment of the method includes lysing thecells packaged with the viruses with an alkaline lysis solution followedby precipitating and removing a majority of host cell proteins with asalt solution. Furthermore, the extracted viral particles can beprecipitated with polyethylene glycol (PEG) and purified bychromatography. The methods disclosed herein are suitable for alldifferent serotypes and species of AAV (e.g., adeno-associated virusserotypes 1-12) or AdV (adenovirus species A-G with their respectiveserotypes) or other “naked” viruses within cells after packaged.

Cell Lysis

In accordance with one embodiment of the disclosure, cells thatenclosing AAV or AdV viruses are lysed with an alkaline lysis solution.Preferably, the lysis solution contains compound(s) or reagent(s) thatare buffering a pH of about 9.0 to about 11.5. The lysis solution canalso include a detergent which is preferably a mild detergent or adetergent that contributes to cell lysis but does not decrease the viralactivities.

An “alkaline lysis solution” or more generally “lysis solution”, as usedherein, refers to a solution that contains compound(s) or reagent(s)that maintain a pH higher than 7.0, or alternatively higher than 7.5,8.0, 8.5, or 9.0. The alkaline lysis solution can also include adetergent that is useful for breaking up cells. The detergent ispreferably a mild detergent such as deoxycholate, octyl thioglucoside,octyl glucoside, dodecyl maltoside, octyl thioglucoside, octylglucoside, alkyl Sulfates, polysorbate 20 (Tween-20), Tergitol-typeNP-40, or the mixtures thereof.

The pH and ingredients of the lysis solution can be determined based onthe desired pH and final concentrations of the ingredients in the cellsample once the lysis solution is added to the sample. In oneembodiment, upon addition of the lysis solution to the cell sample, thepH of the sample is preferably at least 8.0, or alternatively at least8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0,11.1, 11.2, 11.3, 11.4 or 11.5. In another embodiment, the pH of thesample is not higher than 12.0, or alternatively not higher than 11.9,11.8, 11.7 or 11.6. In some embodiments, the pH of the sample is fromabout 8.5 to about 12.0, or from about 9.0 to about 12.0, or from about9.0 to about 11.5, or from about 8.5 to about 11.5, or from about 9.0 toabout 11.0, or from about 9.5 to about 12.0, or from about 9.5 to about11.5, or from about 9.5 to about 11, or from about 9.5 to about 10.5, orfrom about 9.5 to about 10.0, or from about 10.0 to about 12.0, or fromabout 10.0 to about 11.5, or from about 10.0 to about 11.0, or fromabout 10.0 to about 10.5, without limitation. The pH of the lysissolution can be achieved or adjusted with a compound or reagent, like abase, having a concentration from about 0.05 M to about 0.2 M, forinstance.

In some embodiments, the sample has a final concentration of thedetergent at about 0.1% to about 1%, and thus the concentration of thedetergent in the lysis solution can be higher than this. In oneembodiment, the final concentration of the detergent in the sample is atleast about 0.1%, or alternatively at least about 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, or 0.9%. In another embodiment, the finalconcentration of the detergent in the sample is not greater than about1.5%, or alternatively not greater than about 1.4%, 1.3%, 1.2%, 1.1%,1.0%, 0.9%, 0.8%, 0.7%, 0.6% or 0.5%. In some embodiments, the finalconcentration of the detergent in the sample is from about 0.1% to about1.5%, from about 0.1% to about 1.0%, from about 0.2% to about 0.9%, fromabout 0.3% to about 0.8%, or from about 0.4% to about 0.7%, withoutlimitation.

The detergent used is preferably mild unlike strong detergents such asTriton X-100 or sodium dodecyl sulfate (SDS). Non-limiting examples ofdetergents suitable for use here include deoxycholate, octylthioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside,octyl glucoside, alkyl Sulfates, polysorbate 20 (Tween-20), andTergitol-type NP-40. In one embodiment, the detergent used isdeoxycholate, such as sodium deoxycholate (DOC).

In addition to the detergent, the lysis solution can also includecompound ingredients such as N-cyclohexyl-3-aminopropanesulfonic acid(CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassiumcarbonate, sodium phosphate, potassium-phosphate or the combinationsthereof.

Salting Precipitation

It is discovered herein that the cell lysis methods disclosed here,without the need of freeze/thaw cycles, can achieve fairly completelysis of the cells while maintaining high infectivity of the viruses.Such a lysed viral sample can then be subject to a salting precipitationprocess which removes a majority of the contaminating host cellproteins.

The salting precipitation step of the process, in one embodiment,includes the addition of a salting solution that has a neutral or acidicpH (e.g., about 3.5 to about 7.0) and includes a salt at a concentrationthat is at least 0.5 M. A “salting solution” as used herein refers to asolution with the desired pH and salt at a suitable concentration asdisclosed herein. Like for the lysis solution, the pH and saltconcentration of the salting solution can be determined based on thedesired pH and salt concentration in the viral sample and the startingcondition of the sample before the salting solution is added.

In one embodiment, the pH of the viral sample, upon addition of thesalting solution, is not higher than about 7.0, or alternatively nothigher than about 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, or 6.1. Inanother embodiment, the pH of the viral sample, upon addition of thesalting solution, is higher than about 4.0, or alternatively higher thanabout 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, or 5.5. In some embodiments, the pH of the viral sample, uponaddition of the salting solution, is from about 3.5 to about 7.0, fromabout 3.6 to about 7.0, from about 4.1 to about 6.5, from about 4.5 toabout 6.5, from about 5.0 to about 7.0, from about 5.0 to about 6.5, orfrom about 5.0 to about 6.0.

The salt or salts used in the salting solution can be selected from oneor more of the following: sodium acetate, potassium acetate, or sodiumphosphate, without limitation. Final concentration of the salt in theviral sample can be from about 0.5 M and up. In one embodiment, thefinal concentration of the salt or salts is at least 0.5 M, oralternatively at least about 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M,1.2 M, 1.3 M, 1.4 M, or 1.5 M. In one embodiment, the finalconcentration of the salt or salts is not greater than about 3.0 M, 2.5M, 2.0 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M, 1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M,or 1.0 M. In some embodiments, the final concentration of the salt orsalts is from about 0.5 M to about 3.0 M, from about 0.5 M to about 2.5M, from about 0.5 M to about 2.0 M, from about 0.5 M to about 1.5 M,from about 0.5 M to about 1.0 M, from about 1.0 M to about 1.5 M, fromabout 1.0 M to about 2.0 M, or from about 1.0 M to about 2.5, withoutlimitation.

Once the salting solution is added to the viral sample, the majority ofhost cell proteins can be precipitated and removed from the sample bycentrifugation, while the viral particles stay in the supernatant, whichcan be further purified.

Viral Particle Precipitation and Further Purification

The salting step can remove a majority of the proteins from the sampleresulting in viral particles enriched in the sample with little proteincontamination. Such a sample can be further purified as needed, just asremoving the salt in the sample. In one embodiment, the sample is mixedwith a precipitation solution that includes a polyethylene glycol (PEG)at a suitable concentration.

The term “precipitation solution” as used herein generically refers to asolution that includes polyethylene glycol (PEG) which, when added to aviral sample, is able to precipitate viral particles. The molecularweight of the PEG is typically from about 3000 to about 15000, orpreferably from about 3000 to about 10000. The concentration of the PEGin the precipitation solution is, in one embodiment, at least about 5%.Like for the lysis solution and the salting solution, the concentrationof the PEG in the precipitation solution can be determined by thedesired PEG concentration in the viral sample upon addition of theprecipitation solution in the viral sample.

In one embodiment, the final PEG concentration in the viral sample is atleast 5%, or alternatively at least about 6%, 7%, 8%, 9%, 10%, 11% or12%. In another embodiment, the final PEG concentration in the viralsample is not greater than 15%, or alternatively not greater than 14%,13% or 12%.

The PEG in the viral sample can precipitate the viral particles whichcan then be resuspended in a suitable solution.

In one embodiment, the viral sample or resuspended viral particles canbe subject to chromatography. Non-limiting examples of chromatographyinclude silica-based chromatography, resin chromatography or ionexchange chromatography columns. Conditions for using the chromatographycan be set with conventional knowledge in the art.

The lysis, extraction and purification processes described here, in someembodiment, do not require (but do not exclude) certain techniquescommonly used in the art. For instance, in some conventional methods,organic solvents are employed for lysing the cells or degradingproteins. No organic solvents, however, are required by the currenttechnology.

Another commonly used method includes freeze/thaw cycles, which are alsonot required by the present disclosure technology. Yet another methodincludes ultracentrifugation, which typically employs a gradient made ofcesium chloride (CsCl) or iodixanol. Ultracentrifugation or theassociated gradient reagents are also not required in the presenttechnology. Also not required is affinity chromatography, in someembodiments.

Purified Viral Compositions

Viral particles that are prepared by methods of any embodiment of thedisclosure are also provided. It is readily appreciated that in additionto the improved efficiency of viral extraction and purification of thepresent technology, the end product of the technology is also superiorto what can be obtained with the conventional technologies.

For instance, since the present technology does not requireultracentrifugation, the end product would necessarily not contain anycontamination of the gradients, such as cesium chloride (CsCl) oriodixanol.

Also, superior to the affinity chromatography method which is noteffective in removing empty viral particles from viruses, the endproduct of the present technology would contain a much lower amount ofempty particles. In one embodiment, therefore, the end product containsfewer than 1 empty particle per 10,000 viral particles, or alternativelyfewer than 1 empty particle per 20,000, 50,000, 100,000, 500,000, 10⁶,10⁷, 10⁸, 10⁹, or 10¹⁰ viral particles.

Another difference between the product of the present process and thosemade by conventional technologies is that the present product has lessprotein contamination. In one embodiment, the end product contains lessthan about 1/10,000 (w/w) host cell proteins over the total amount ofthe viral particles, or alternatively less than about 1/20,000, 1/50,0001/100,000, 1/500,000, or 1/1,000,000 (w/w) host cell proteins.

Yet another advantage of the present technology is that the purifiedviruses retain greater infectivity as compared to what has been done inthe prior art. In one embodiment, for each 10¹³ of the particles in aprepared sample, there are at least 10¹⁰ transducing units of activeviruses. In another embodiment, for each 10¹³ of the particles in aprepared sample, there are at least 1×10⁹, 2×10⁹, 5×10⁹, 2×10¹⁰, 5×10¹⁰,1×10¹¹, 2×10¹¹, 5×10¹¹, or 1×10¹² transducing units of viruses.

Methods and Compositions

Adeno-associated virus (AAV) is a small virus which infects humans andsome other primate species. AAV is not currently known to cause disease.The virus causes a mild immune response, lending further support to itsapparent lack of pathogenicity. Gene therapy vectors using AAV caninfect both dividing and quiescent cells and persist in anextrachromosomal state without integrating into the genome of the hostcell, although in the native virus some integration of virally carriedgenes into the host genome does occur. These features make AAV anattractive candidate for creating viral vectors for gene therapy, andfor the creation of isogenic human disease models.

The best studied AAV serotype is serotype 2. Serotypes 2, 3, 5, and 6were discovered in human cells, AAV serotypes 1, 4, and 7-11 in nonhumanprimate samples. There have been 11 AAV serotypes described. AAV capsidproteins contain 12 hypervariable surface regions, with most variabilityoccurring in the threefold proximal peaks, but the parvovirus genome ingeneral presents highly conserved replication and structural genesacross serotypes. All of the known serotypes can infect cells frommultiple diverse tissue types. Tissue specificity is determined by thecapsid serotype and pseudotyping of AAV vectors to alter their tropismrange will likely be important to their use in therapy.

Adenoviruses (AdV) are medium-sized (90-100 nm), nonenveloped (withoutan outer lipid bilayer) viruses with an icosahedral nucleocapsidcontaining a double stranded DNA genome. They have a broad range ofvertebrate hosts; in humans, more than 50 distinct adenoviral serotypes,belonging to seven species (A-G), have been found to cause a wide rangeof illnesses, from mild respiratory infections in young children (knownas the common cold) to life-threatening multi-organ disease in peoplewith a weakened immune system.

Methods of preparing recombinant AAV and AdV and packaging them intosuitable host cells are known in the art. For AAV, viral productioncells can include at least the minimum components required to generateAAV particles, where production of an AAV DNase resistant genomecontaining particles involves packaging an expression cassette into AAVcapsids. The minimum required components include, for instance, anexpression cassette to be packaged into the AAV capsids, AAV cap, andAAV rep or functional fragments thereof, and helper functions.

Suitable cells and cell lines have been described for use in productionof AAV and AdV. The cells themselves may be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells and eukaryoticcells including insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, A549, WEHI, 3T3, 10T1/2, BHK,MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293cell, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast,hepatocyte and myoblast cells derived from mammals including human,monkey, mouse, rat, rabbit, and hamster.

Generally, the expression cassette is composed of, at a minimum, a 5′AAV inverted terminal repeat (ITR), a nucleic acid sequence encoding adesirable therapeutic, immunogen, or antigen operably linked toregulatory sequences which direct expression thereof, and a 3′ AAV ITR.In one embodiment, the 5′ and/or 3′ ITRs of AAV serotype 2 are used.However, 5′ and 3′ ITRs from other suitable sources may be selected. Itis this expression cassette that is packaged into capsid proteins toform an AAV virion (particle).

In addition to the expression cassette, the cells contain the sequenceswhich drive expression of AAV capsids in the cells (cap sequences) andrep sequences of the same source as the source of the AAV ITRs found inthe expression cassette, or a cross-complementing source. The AAV capand rep sequences may be independently selected from different AAVparental sequences and be introduced into the host cell in a suitablemanner known to one in the art. While the full-length rep gene may beutilized, it has been found that smaller fragments thereof, i.e., therep78/68 and the rep52/40 are sufficient to permit replication andpackaging of the AAV.

The cells also require helper functions in order to package the AAV ofthe invention. Optionally, these helper functions may be supplied by aherpesvirus. In another embodiment, the necessary helper functions areeach provided from a human or non-human primate adenovirus source, suchas are available from a variety of sources, including the American TypeCulture Collection (ATCC), Manassas, Va. (US).

The present disclosure also provides, in certain embodiments,compositions and methods for using the purified viruses as a therapeuticor vaccine agent.

EXAMPLES

The following examples are included to demonstrate specific embodimentsof the disclosure. It should be appreciated by those of skills in theart that the techniques disclosed in the examples which follow representtechniques to function well in the practice of the disclosure, and thuscan be considered to constitute specific modes for its practice.However, those of skills in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

Example 1: Lysis, Extraction and Purification of Adeno-Associated Virusand Adenovirus from Host Cells

This example tests a process for lysing, extracting and purifying AAVand AdV viral particles from their packaged cells (host cells). Theexample demonstrates that this process achieves higher recovery andpurity of viral particles than conventional methods. In addition, thisprocess is easy to implement and can be scaled up readily.

Methods and Materials

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) andcell culture dishes were purchased from Fisher Scientific. 6-well and12-well cell culture plates were purchased from Santa CruzBiotechnology. Plasmid DNA Maxiprep kit was purchased from Qiagen.Lipofectamine 2000 Transfection Reagent was purchased from LifeTechnologies. DNase I, Maxima Sybr Green qPCR Master Mix (2×), PierceBCA Protein Assay kit, SDS-PAGE minigels, Amicon Ultra-4 CentrifugalFilter and all chemicals were purchased from Fisher Scientific.Silica-based chromatography columns were purchased from AgilentTechnologies, Inc. and Bonna-Agela Technologies Inc.

Cell Culture

HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM)containing 10% Fetal Bovine Serum (FBS) in a 37° C. incubator with 5%CO₂ and subcultured every 3-5 days. Cells were split 1:2 in 15-cm dishesfor transfection for AAV packaging or for infection for AdVamplification. Cells were split onto 6-well or 12-well cell cultureplates from infection testing.

AAV Packaging

HEK293 cells were split onto 15-cm dishes and transfected on the 2^(nd)day using Lipofectamine 2000 Transfection Reagent or calcium phosphateprecipitation method. Plasmid DNA used for AAV packaging includedGFP-recombinant AAV viral vector and AAV packaging plasmids. Afterovernight incubation, the medium was replaced with fresh growth mediumand the transfected cells were incubated for additional two days beforecollecting cells by centrifugation at 2000 rpm for 5 minutes. TheAAV-packaged cells were used for viral extraction immediately or storedat −80° C. until use. The medium was saved for PEG precipitation ofviral particles within it. PEG precipitation was conducted using 6-8%PEG8000 and 1-hour incubation at 4° C., followed by centrifugation at10,000 rpm for 10 minutes. The PEG precipitates were suspended in PBS byvortexing and pipetting followed by clarifying at 5000×g for 5 minutes.The viral suspensions were used in pH, detergent and salt testing asshown in FIG. 1 and FIG. 3 panel A.

AdV Generation

HEK293 cells were split onto a T75 flask and transfected with aGFP-recombinant adenoviral plasmid plus Lipofectamine 2000. The mediumwas replaced with fresh growth medium 24 hours post transfection. Cellswere incubated for a week before harvesting cells. Cells were suspendedin PBS and extracted by freeze/thaw 3 times, followed by clarifying at5000×g for 5 minutes. One third of supernatant was used for infection ofa T175 of HEK293 cells for further viral production and the rest wasstored as primary stock. The infected cells were collected after a weekof incubation and the viral particles were extracted by freeze/thaw 3times as stock at −80° C. until use for viral production. For testingassays, 15-cm dishes of HEK293 cells were incubated for 3-5 days afterinfection with AdV virus until >90% cells were cytopathic. Cells werethen collected by centrifugation at 2000 rpm for 5 minutes. Cell pelletswere used for viral extraction immediately or stored at −80° C. untiluse. The medium was saved for PEG precipitation of viral particleswithin it. PEG precipitation was conducted as the same as describedabove for AAV and viral suspensions were used in pH, detergent and salttesting.

Condition Testing

AAV and AdV viral solutions were mixed with an equal volume of pHbuffered solutions, detergent solutions or salt solutions as indicatedin figures and incubated at room temperature for 2 hours before used forinfection in HEK293 cells. Infectivity visualized by GFP wasinvestigated to estimate the viral activity. Determination of the rangesof pH value and amounts of detergents suitable for AAV and AdV virusesto maintain their activities is aimed to design cell lysis solutions toeasily release the viral particles from their packaging cells.Determination of the viral suitability to a range of salt solutions isexpected to learn the possibility to employ salting precipitation in theviral purification procedures.

Viral Purification

Lysis of host cells is an important step that enabled rapid lysis of theviral host cells and complete release of the viral particles. Based onthe results from condition testing that AAV and AdV viruses were able tomaintain their activities in a wide range of pH values and certainamounts of detergents such as sodium dodecyl sulfate (SDS) and sodiumdeoxycholate (DOC), a serial of lysis solutions were designed, includingvarious acidic solutions and alkaline solutions without or with additionof detergents as shown in Table 2 and 3. After the discovery thatalkaline solutions with addition of detergents such as DOC or DOC plusSDS achieved effective lysis of AAV- and AdV-packaged cells, variousalkaline lysis solutions were further prepared and tested as shown inTable 4 and 5. Suitable detergents used here could also includedeoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside,octyl thioglucoside, octyl glucoside, alkyl Sulfates, Polysorbate 20(Tween-20), Tergitol-type NP-40 and their combinations. In process, AAV-or AdV-packaged cells were collected by centrifugation at 2000 rpm for 5minutes and then lysed in the above lysis solutions. Cells were lysedwith radioimmunoprecipitation assay (RIPA) buffer [25 mM Tris-Cl, pH7-8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate (DOC), 1% NP-40 andcocktail protease inhibitors] as total protein control or extractedusing 3 cycles of freeze in −80° C. and thaw in a 37° C. water bath ascomparative control.

As an extraction step, we first tested the possibility to use saltingprecipitation to remove the host cell proteins but retain the viralparticles in supernatants. After testing the tolerance of AAV and AdVviruses to salt as described above in which we found that both of theviruses display wide suitability to high salt, we prepared serials ofvarious salt solutions (Table 6) for test of salting precipitation inthe viral extraction following cell lysis. Salt solutions were added tocell lysates and were mixed quickly. After salting precipitationoccurred, the mixed solutions were clarified by high-speedcentrifugation and removed the precipitates. Supernatants were used forinfection in HEK293 cells to validate viral particles remaining insolution. It was demonstrated that salting precipitation process removedthe majority of impurities of host cell proteins and efficientlyretained the viral particles in solution, the extracted viral solutionsconsiderably cleaner that could be further processed with PEGfractionation or directly used for further purification by columnchromatography.

PEG fractionation was then tested to desalt, enrich and further purifythe extracted viral solutions following salting process. Various sizesof PEG with average molecular weights from 3000 to 10000 were tested bymaking stock solutions and adding different amounts to the viralsolutions. After mixing, the mixtures were incubated on ice for 0.5-2hours and then centrifuged at a high speed for 15 minutes to precipitatethe viral particles. The viral particles were resuspended in PBS forinfection to validate the viruses or in an appropriate buffer forfurther purification by column chromatography. It was found that PEGfractionation led to high-purity viral solutions that could be suitablein use for many research purposes.

The next step was column chromatography purification of the AAV and AdVparticles, followed by concentration of the viral solutions withcentrifugal filters, in order to yield high-titer, ultrapure viralsolutions. AAV or AdV viral particles, either the extracted viralsolutions from salting process or the resuspended viral solutions fromPEG fractionation, were applied onto the chromatography columns thatwere silica-based chromatography, resin chromatography or ion exchangechromatography columns. Serials of viral suspension buffers, columnwashing buffers and elution buffers were prepared and optimized fordifferent chromatography columns. The viral eluents were concentratedusing Amicon Centrifugal Filters or other centrifugal filters.

Infectivity Assay

Infection in HEK293 cells was performed as major means of investigationof viral activities in developing the present techniques forpurification of AAV and AdV particles. Cells were split in 12-well or6-well plates with 80-90% confluence in DMEM containing 5% FBS per wellone day before use for infection. Amount of viral solutions were addedto each well according to viral concentrations, ionic strength and pHvalue of the viral solution, ranged about 1-10 μl per well or dilutedbefore used for infection. Since all viruses used are GFP-contained, theinfectivity was investigated by viewing GFP cells that were photographedand counted under a fluorescence microscope. Photographs were takenunder a 4× lens and GFP cells were counted in fields of a 10× lens. AdVinfectivity was investigated in 2-3 days after infection while AAVinfectivity was investigated in 3-5 days. It was important that GFPcells after infection were countable in fields of a 10x lens. If numbersof GFP cells were too high or too low to count, infection was repeatedwith adjusted amounts of viral solutions or with new viral solutionsfrom repeated experiments.

qPCR Titration

AAV and AdV viral solutions after chromatography column purificationwere titrated using quantitative real-time PCR (qPCR). The viralsolutions were first treated with DNase I in 10 μl reaction mixturecontaining 5 units of DNase I and then titrated by means of qPCR in 25μl reaction mixture containing 2× Sybr Green qPCR Master Mix and onepair of primers as listed in Table 1 using a standard qPCR cyclingparadigm. Serials of 10-fold dilutions of viral vectors were used asstandards in the qPCR titration. Titers of viral solutions werecalculated as viral genomic copies per milliliter (ml) using thestandard curves.

TABLE 1 Primers were used in qPCR for AAV viral titration. Ampli-Titration Target ID Sequences (SEQ ID NO: _) con virus GFP Fw_GFP: 76 bp AAV or 5′-TCTGCACCACCGGCAAGCTGC-  AdV 3′ (SEQ ID NO: 1) Rv_GFP:5′-GAGAAGCACTGCACGCCGTAG-  3′ (SEQ ID NO: 2) hGH_PA Fw_pA: 102 bp AAV5′-GGTCTCCAACTCCTAATCTCAG-  3′ (SEQ ID NO: 3) Rv_pA:5′-AAAATCAGAAGGACAGGGAAGG-  3′ (SEQ ID NO: 4) CMV Fw_CMV: 128 bp AdV5′-TTCCTACTTGGCAGTACATCTACG-  3′ (SEQ ID NO: 5) Rv_CMV:5′-GTCAATGGGGTGGAGACTTGG-  3′ (SEQ ID NO: 6)BCA Assay

Protein amounts contained in viral solutions were determined usingbicinchoninic acid assay (BCA assay) following the BCA Protein AssayKit's protocol. Briefly, the assays were conducted by adding 25 μlsample solutions and 200 μl BCA solution (mixed A and B, 50:1) per wellin 96-well plates. The plates were incubated at 37° C. for 30 minutes,followed by reading at 562 nm using a plate reader. Bovine serum albumin(BSA) was used to make a standard curve with serial dilutions from astock of 10 mg/ml. To lower influence from higher salt concentration insolutions after salting precipitation, two strategies were used: (1) Thehigh salt samples were diluted 5 folds in H2O, so that 5 μl sample plus20 μl H2O was added per well for BCA assay; (2) A similar salt solution(salt mix) was prepared and 5 μl of the salt mix was added to each wellof those of low salt samples including standard dilutions to reduce theinfluence of high salt in BCA assay.

SDS-PAGE Electrophoresis

Protein amounts contained in viral solutions were visualized by means ofSDS-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Viralsolutions from alkaline lysis/salting precipitation were diluted 4 foldwith H₂O and then mixed with 5×SDS sample buffer. Sample solutions fromfreeze/thaw and RIPA lysis were mixed with 5×SDS sample buffer and thendiluted with 1×SDS sample buffer. All samples were portioned in the samevolume based on original cell suspensions and same volume of each samplewas loaded onto 10% SDS gels. Gels were run at 140 Voltages for about 1hour until the dye bromophenol blue reached at the gel's bottom. Afterelectrophoresis, gels were fixed in a fixing solution containing 50%(v/v) methanol in water with 10% (v/v) acetic acid before stainingovernight in a Coomassie Blue Staining solution containing 50% (v/v)methanol in water with 10% (v/v) acetic acid and 0.25% (w/v) CoomassieBlue R-250. Destaining was performed in a destaining solution containing50% methanol and 10% acetic acid. After completely destaining, gels werekept in 5% (v/v) acetic acid. Gels were scanned into image files using ascanner, of which density of gels were quantified using NIH Image J.

Results

Adeno-Associated Virus and Adenovirus Maintain their Infectivity in aWide Range of pH Values from about 3.6 to about 11.5.

This example first tested the suitability of AAV and AdV to a wide rangeof pH values from about 3.6 to about 11.5 in order to determine pHvalues for development of desirable cell lysis solutions. Viralsolutions were incubated with the indicated pH solutions for 2 hours atroom temperature before used for infection in HEK293 cells. Throughinvestigation of their GFP expression, we found that AAV maintained itsinfectivity at all tested pH values from pH 3.6 to pH 11.5 and AdV wasstable to maintain its infectivity at the pH values from pH3.6 to pH10.5but unstable over pH 11.0 (FIG. 1 panels A and B). In contrast, LtV wasused for comparison, which has a narrow range of pH values from pH 6.0to pH 9.5 to maintain its infectivity (FIG. 1 panels A and B). Theseresults, therefore, suggest determining the desired pH values of celllysis solutions from pH 3.6 to pH 11.5 for AAV and from pH 3.6 to pH10.5 for AdV that mostly conduce to lysis of cells and do not obviouslyaffect the viral activity.

Adeno-Associated Virus and Adenovirus Maintain their Infectivity in aRange of Amounts of Sodium Deoxycholate from about 0.1% to about 1.0%,but are Sensitive to Sodium Dodecyl Sulfate.

To test the possibility to use detergents in cell lysis solutions forlysis of AAV- and/or AdV-packaging cells, we determined the tolerance ofthese viruses to detergents including sodium dodecyl sulfate (SDS),Triton X-100 and sodium deoxycholate (DOC) that are commonly used forcell lysis. Viral solutions were incubated with the indicated detergentsfor 2 hours before used for infection. HEK293 cells are very sensitiveto Triton X-100 but not to SDS and DOC in amounts used in the testing,implicating that Triton X-100 may be toxic to the viruses. SDS treatmentwas stringent to both viruses, which shows that AdV is more sensitive,even 0.1% SDS abolished all viral infectivity but AAV infectivity wasalso dramatically decreased with increased SDS concentrations, 30% downat 0.1% SDS and 50% down at 0.5% SDS (FIG. 1 panels C and D). Incontrast, both viruses maintain high infectivity in a range of DOCamounts from 0.05% to 1.0% tested (FIG. 1 panels C and D). It istherefore suggested from these results that DOC, but neither SDS norTriton X-100, is the best candidate of detergent for cell lysissolutions because of it's compatibility with both AAV and AdV viruses.

Alkaline Solutions, but not Acidic Solutions, Partially Lyse Viral HostCells

According to suitable pH values determined above, we prepared severalacidic solutions and alkaline solutions (Table 2) to test whether HEK293cells—the viral packaging cells could be lysed under acidic and/oralkaline conditions. Cells were collected and resuspended in thesesolutions respectively. After mixing, we found that cells-suspendedalkaline solutions were much clearer than cells-suspended acidicsolutions, the former with a little of pellets out while the later withmuch more pellets out after centrifugation at 5000 rpm for 2 minutes(Table 2). It is obviously indicated that alkaline solutions withpH9.0-11.5 partially, but not completely, lyse the viral host cellswhile acidic solutions with pH3.6-5.0 did not lyse the cells. Thus, itis concluded that these alkaline solutions, although better than acidicsolutions, are not capable to completely lyse the viral host cells.

TABLE 2 HEK293 cells are partially lysed in alkaline, but not acidic,solutions. Cell lysis Spin Lysis solutions (5000 rpm, Type Component pHVisible 2 min) Acidic HCl, 0.1-0.5N  ~1 Not lysed Pellet out NaAc,0.1-0.5M 3.6-5.0 Not lysed Pellet out Alkaline NaOH, 0.1-0.5N ~13 LysedNo pellet Glycine/Na,  9.0-11.5 Not fully Lysed Small pellet 0.1-0.5MDetergents Enable Alkaline Solutions to Efficiently Lyse AAV and AdVPackaging Cells.

As determined above, AAV and AdV were sensitive to SDS but compatible toDOC (FIG. 1 panels C and D). Therefore, we tested whether DOC or DOCplus SDS improves cell lysis by adding 0.1%-1.0% DOC with or without0.01%-0.2% SDS to the above alkaline solutions and found that additionof the detergents led to complete lysis of cells in the pH range of pH9.6-11.5 tested (Table 3). Other detergents, including deoxycholate,octyl thioglucoside, octyl glucoside, dodecyl maltoside, octylthioglucoside, octyl glucoside, alkyl Sulfates, Polysorbate 20(Tween-20), Tergitol-type NP-40 and their combinations, may be alsosuitable to conduce to cell lysis. Therefore desirable alkaline lysissolutions are suggested that contain reagents or compounds withbuffering capability of pH9.0-11.5 and appropriate amounts of detergentssuch as DOC and SDS.

TABLE 3 Sodium deoxycholate enables alkaline solutions to completelylyse HEK293 cells. Alkaline solutions Cell lysis Alkaline solutions pHDOC SDS Visible Spin 1 Spin 2 NaOH, 0.1-0.5N ~13 0.1%-1.0% No Lysed Nopellet No pellet 0.1%-1.0% 0.01%-0.5% Lysed (5K rpm (10K rpm 2 min) 2min) Glycine/Na, 0.1-0.5M 90-11.5 0.1%-1.0% No Lysed No pellet No pellet(5K rpm (10K rpm 0.1%-1.0% 0.01%-0.5% Lysed 2 min) 2 min)Multiple Alkaline Solutions Effectively Lyse HEK293 Cells with Additionof Detergents.

We further investigated cell lysis with different kinds of alkalinesolutions containing detergent DOC as example as listed in Table 4 andfound all these kinds of alkaline solutions are efficient in lysis ofHEK293 cells. Next, we chose 7 kinds of alkaline solutions as listed inTable 5 to lyse AAV- and AdV-packaged cells and investigated viralinfectivity after lysis. It is known that freeze/thaw is most commonlyused to extract AAV and AdV particles, so we extracted AAV or AdVparticles from their packaged cells using freeze/thaw three times inparallel as comparative control. Viral infectivity was determined byinvestigation of GFP-expressed cells that were photographed (FIG. 2panel A) and counted (FIG. 2 panel B). We found that these alkalinesolutions are all effective in lysing the host cells and release bothAAV and AdV viral particles that displayed higher infectivity than thoseextracted by freeze/thaw (FIG. 2 panels A and B). Consistent to theresults from pH testing above, AdV infectivity was much decreased in thealkaline lysis solutions with pH11.0 (FIG. 2 panels A and B). It istherefore concluded that alkaline solutions plus appropriate amounts ofdetergents such as DOC are sufficient to lyse AAV- and AdV-packagedcells and completely release the viral particles, the pH9.0 to 11.5 forAAV and the pH9.0 to 10.5 for AAV. This new cell lysis method canovercome the shortages of freeze/thaw method including time-consumingand viral activity-decreasing.

TABLE 4 Multiple alkaline solutions with DOC effectively lyse HEK293cells. Alkaline lysis solutions Chemicals Concentration pH Cell lysisNa₂HPO₄ 0.1-0.5M ~9 Good K₂HPO₄ 0.1-0.5M ~9 Good Glycine/NaOH 0.1-0.5M 9.5-10.5 Good Tris base 0.1-0.5M ~10  Good CAPS/NaOH 0.1-0.5M 10.0-11.5Good Na₂CO₃/NaOH 0.1-0.5M 10.5-11.0 Good Na₂HPO₄/NaOH 0.1-0.5M 10.5-11.0Good

TABLE 5 Alkaline solutions are used for lysis of virus-packaged cells.Alkaline lysis solutions Viral activity No. Chemicals pH AAV AdV 1Na₂HPO₄ 8.9 Good Good 2 Glycine/NaOH 9.6 Good Good 3 10.0 Good Good 410.5 Good Good 5 CAPS/NaOH 10.0 Good Good 6 11.0 Good No 7 Na₂HPO₄/NaOH11.0 Good NoSalting Precipitation is Suitable to Precipitate Protein Impurities andRetain AAV and AdV Viral Particles in Solutions.

Massive host cell proteins exist in viral solutions as their packagedcells are lysed. How to remove these protein impurities is the majorconcern in these viral purifications. It is known that saltingprecipitation is a potent technique commonly used in protein chemistry.To test the possibility using salting precipitation to remove proteinsin viral solutions, we investigated the tolerance of AAV and AdV to highsalt solutions and found that both AAV and AdV maintain highlyconsistent infectivity after incubation with solutions of salt up to 2.0M NaCl, while LtV, in contrast, shows less tolerance to high saltsolutions (FIG. 3, panels A). These results implicate the possibilitythat salting precipitation could be used to remove protein impurities inthese viral solutions.

In test of salting precipitation, we lysed AAV- and AdV-packaged cellsin the 7 kinds of alkaline lysis solutions as listed in Table 5, tothese cell lysates 0.5-3 M NaAc, pH 5.5 was added and mixed quickly. Asexpected, large precipitation occurred. The precipitates were removed bycentrifugation and the supernatants were saved for infection. Results ofviral infectivity show that both kinds of the viral particles perfectlyremain in the supernatants after salting precipitation (FIG. 3 panels Band C). Importantly, we found that the new method using alkalinelysis/salting precipitation produced AAV and AdV particles 20-40% higherthan those extracted by freeze/thaw (FIG. 3 panels B and C), suggestingthat alkaline lysis is more efficient than freeze/thaw to release AAVand AdV viral particles from their host cells.

Salting Precipitation Removes the Majority of Host Cell Proteins fromAAV and AdV Solutions.

To determine the effectivity of salting precipitation, we prepared twosets of samples: Set 1 used normal HEK293 cells and Set 2 usedAAV-packaged HEK293 cells, and lysed these cells with alkaline lysissolutions followed by salting precipitation. After centrifugation toremove out precipitates, protein concentrations in the supernatants weremeasured by BCA assay (FIG. 4 panel A) and SDS-PAGE electrophoresis(FIG. 4 panel B). Both results consistently display that the majority ofprotein impurities in all the cell lysates was removed by salting out incomparison with whole cell lysate (RIPA buffer lysed) and extract byfreeze/thaw (FIG. 4 panels A and B). Furthermore, a trend is observedthat higher pH of alkaline lysis solutions led to more proteins removal(FIG. 4 panels A and B).

Investigation of salting-out effectiveness of various salt solutionsdiscovered a variety of salts are suitable for salting precipitation toprecipitate impurities out and retain the viral particles in solutionsexcept ammonium salts that inhibit the infectivity of both AAV and AdVviruses (Table 6). Besides, salt doses and pH values of salt solutionswere further demonstrated that protein removal by salting precipitationis salt dose dependent and can be shifted up with lower pH (FIG. 4 panelC).

TABLE 6 Various salt solutions and their effectiveness in saltingprecipitation Salt solution Effectiveness Chemicals Concentration pHProtein removal Viral activity NaH2PO4 1.0-4.0M 4.0-6.0 Good Good NaAc1.0-4.0M 4.0-6.0 Good Good KAc 1.0-4.0M 4.0-6.0 Good Good NH4Ac 1.0-6.0M4.0-6.0 Good Inhibited NaCl 1.0-4.0M 4.0-6.0 Good Good KCl 1.0-4.0M4.0-6.0 Good GoodPolyethylene Glycol Fractionation Desalts and Enriches AAV and AdVParticles

The next step attempted to separate the viruses from the high saltsolutions either using column chromatography as described below orthrough fractionation of viral particles into a fraction of solution orprecipitate with organic solvents or a polymer such as polyethyleneglycol (PEG). In test of desalting, viral particles of both AAV and AdVwere found able to be fractionated in a two-phase system includingNa—HPO4/PEG or K—PO4/PEG or precipitated by organic solvents such asacetone and isopropanol. These methods, however, lead to decrease theviral infectivity (data not shown). Use of various molecular sizes ofPEG from PEG3000 to PEG 10000 showed effective fractionation of the twokinds of viruses from the high salt solutions into precipitates (datanot shown). For example as shown in FIG. 5 panels A and B, variousamounts of PEG8000 were able to precipitate both AAV and AdV viralparticles that were resuspended in PBS. Results from infection show thatPEG fractionation yielded an extremely high recovery of the two kinds ofviruses (FIG. 5 panel C) and its selectivity for precipitation of theviral particles leading to further removal of contaminant proteins (FIG.5 panel D). It is, therefore, concluded that PEG fractionation is anexcellent step that desalts, enriches and further purifies the viralparticles following alkaline lysis/salting precipitation processes.Importantly, the viral solutions of AAV or AdV resulted from PEGfractionation are high-quality viral preparations that can be used formany research purposes.

Various Kinds of Column Chromatography Yield Ultrapure Viral Particlesof AAV and AdV.

Although the viral solutions resuspended from PEG fractionation are inhigh quality and suitable for research purposes, further efforts weremade to increase the purity of the viral particles in order to meet therequirements in use for all research purposes and/or preclinical orclinical trials. A desired choice of technique is a kind of columnchromatography that is an easy, efficient and scalable technique. Intesting, we found that various kinds of column chromatography are ableto further purify both AAV and AdV viral particles, includingsilica-based chromatography columns, resin and ion exchangechromatography columns (Data not shown). As example shown in FIG. 6, akind of cation exchange columns were used to purify AAV or AdV particlesfrom viral solutions extracted using alkaline lysis/saltingprecipitation as described in FIG. 3 (panel A) and silica-basedchromatography columns were used to purify AAV or AdV particles fromviral resuspensions of PEG precipitates as described in FIG. 5 (panelB). After concentration of the eluted viral solutions from thesecolumns, the resulting viral solutions were titrated using qPCR, whichshow high titers about 10¹³ GC/ml of AAV or higher than 10¹³ GC/ml ofAdV (FIG. 6 panel C).

The whole procedure we present here for purification of AAV and AdVviral particles include 3 continuous steps to remove protein impurities.After cell lysis using an alkaline lysis, salting precipitationprocessed using a high salt solution is the first step that removes themajority of host cell proteins, about 80-90% (FIG. 7 panels A and B).PEG fractionation is the following step that further removes contaminantproteins from the viral particles, about 70-80% (FIG. 7 panels A and B).Chromatography purification is the final step that removes remainingcontaminant proteins and ensures ultrapurity of the viral solutions(FIG. 7 panels C and D) that can be concentrated using a centrifugalfilter to yield a final high-titer viral solution (FIG. 7 panels E-G).The new procedures used for the two viral purifications arecharacterized ultrahigh purity (near 100% viral particles) (FIG. 7panels C, D and G), high recovery (>90%) (FIG. 7 panel E) and high titer(10¹⁰ TU/ml) (FIG. 7 panel F).

The Artistic Approach Exhibits Unparalleled Advantages in Production ofHigh Purity AAV Solution.

A comparative study as example demonstrated the advantages of our newprocedure over commercial products. Three of AAV purification kits thatare marked as Kit A, Kit B and Kit C were found from the current market,commercially available from different companies. These kits were usedfor AAV purification from AAV-packaged cells following the productprotocols, compared with our present procedure as described above(marked as C&M). Results from infection and protein assays with theresulting viral solutions are shown in FIG. 8, in which we first foundthat C&M-purified viral solution displayed much higher infectivity(panel A) and >90% viral recovery, while the viral recovery using KitsA, B and C was 27.88±4.82, 6.36±1.27, 4.52±0.66 respectively (panel B);the titer of C&M-purified viral solution is near 10¹⁰ TU/ml, 3-12 timesover those of viral solutions purified by commercial kits (panel C),while the later contains much more contaminant proteins than the former(panels D and E). Using the viral transducing units (TU) per pg proteinto estimate the viral purity, C&M-purified viral solution shows149.81±4.60 TU/pg protein, the purity 24-68 times over the viralsolutions purified with Kits A-C (FIG. 8 panel F). It is, therefore,concluded that our artistic approach exhibits unparalleled advantages inyielding a high-titer, high-purity AAV solution over the currentcommercial kits.

In summary, this study presents a new approach for purification of AAVor AdV particles from their packaged cells, which includes a fewimportant steps as follows (FIG. 9): (1) cell lysis (cell lysates) usingan alkaline solution that contains compounds with buffering capabilitymaintaining pH value of 9.0-11.5; (2) salting precipitation process byadding a salting solution (high salt) to cell lysates to remove themajority of host cell protein impurities in precipitates and retain AAVor AdV viral particles in solutions (viral extracts); (3) PEGfractionation by adding PEG to viral extracts to separate viralparticles in precipitates that can be resuspended in an appropriatesolution (viral suspensions) and retain protein impurities in solutionleading to desalting, enriching and purifying the viral particles; and(4) column chromatography by using silica-based chromatography columnsto finally purify the above viral suspensions followed by concentrationwith centrifugal filters to produce high-titer, ultrapure viralsolutions. Moreover, this is a very easy, time-saving procedure that canbe finished in about 3 hours and is suitable for purification of allserotypes of AAV or AdV or any other species of packaged virusesretained in host cells. All the solutions used in the procedure do notcontain any toxic compounds or reagents so that the resulting viralsolutions can meet the quality requirements for most research purposesand/or preclinical and clinical trials.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” “containing”, etc., shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art to whichthe disclosure pertains.

The invention claimed is:
 1. A method of extracting viral particles of arecombinant adeno associated virus (AAV) or an adenovirus (AdV) from asample comprising cells enclosing the viral particles, comprising:lysing the cells by the addition of an alkaline lysis solution to thesample such that the sample comprises about 0.1% to about 1% of adetergent comprising a salt of deoxycholic acid and has a pH of about9.5 to about 11 under suitable conditions to lyse the cells; thenprecipitating proteins released from the lysed cells by adding a saltingsolution to the sample such that the sample comprises about 0.5 M toabout 2.0 M of a salt selected from the group consisting of sodiumacetate, potassium acetate and combinations thereof and has a pH ofabout 4.0 to about 7.0; then removing the precipitated proteins toobtain a supernatant comprising the viral particles; and thenprecipitating the viral particles by adding a precipitation solution tothe supernatant such that the supernatant comprises about 5% to about12% of a polyethylene glycol (PEG), wherein the method does not includeuse of ultracentrifugation or freeze-thaw cycles.
 2. The method of claim1, wherein the viral particles are of any one of AAV serotypes 1-11 orany one of AdV species A-G.
 3. The method of claim 1, wherein thedetergent further comprises sodium dodecyl sulfate (SDS).
 4. The methodof claim 1, wherein the alkaline lysis solution has a pH of about 9.5 toabout 11 and comprises, in addition to the detergent, one or morereagents selected from the group consisting ofN-cyclohexyl-3-aminopropanesulfonic acid (CAPS), glycine/sodium, glycinepotassium, sodium carbonate, potassium carbonate, sodium phosphate,potassium-phosphate and combinations thereof.
 5. The method of claim 1,wherein the salting solution does not include an ammonium salt.
 6. Themethod of claim 1, wherein the PEG has an average molecular weight ofabout 3000 to about
 10000. 7. The method of claim 1, further comprisingsubjecting the precipitated viral particles to chromatography.
 8. Themethod of claim 7, wherein the chromatography is silica-basedchromatography, resin chromatography or ion exchange chromatographycolumns.
 9. The method of claim 1, wherein the method does not includethe use of an organic solvent.
 10. The method of claim 1, wherein themethod does not include the use of affinity chromatography.